Abstract: :
Purpose: We previously showed that transgenic mice (tg), Kerapr3.2–Bgn,over–expressing biglycan under the keratocan promoterexhibited exposure keratitis. These mice revealed a differentpremature eye opening phenotype from other failure in eyelidclosure such as eye–open at birth (eob), lidgap–Gates(lgGa), open–eyes (oe), and gaping lids (gp). This studyis to characterize the epithelial–mesenchymal interactionsessential for eyelid morphogenesis and uncover the mechanismby which excess biglycan interrupts this important developmentalprocess.Methods: Developing embryos of tg and non–tg littermateswere evaluated at days 13.5∼ 17.0 of gestation. Light, confocoal,and scanning electron microspcopes were used to examine thepathology in tg. Cell migration assay was used to test if eyelidmesenchymal cell migration can be induced by eyelid epithelium.Neutralizing antibodies against TGF–α, TGF–ßs,FGFs, and BMPs in cell migration assays was used to determinethe identity of the cytokine released by the eyelid epithelium.Ip–western analysis was employed to identify cytokine–biglycancomplex. In situ hybridization and immunostaining were performedto show if autocrine/paracrine loop was altered in tg. Results:The Kerapr3.2–Bgn mice exhibited premature eye openingand exposure keratitis due to perturbation of eyelid muscleformation and the failure of Meibomian glands formation. Invitro analysis revealed that biglycan, and anti–TGF–αand EGFR antibodies, inhibit mesenchymal cell migration inducedby eyelid epithelium, and that biglycan binds to TGF–α.The defects of TGF–α signaling by excess biglycan werefurther augmented by the interruption of the autocrine/paracrineloop of the EGFR signaling pathway of HB–EGF expressionelicited by TGF–α and resulted in eyelid malformation.Conclusions:Excess biglycan sequesters TGF–α and disrupts its morphogenegradient, therefore, results in eyelid malformation. These resultsare consistent with the notion that biglycan serves as a regulatorymolecule for the formation of a TGF–α morphogen gradientessential for eyelid morphogenesis.